This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sphingolipids are becoming well established as signaling molecules in aging, cell proliferation, disease states such as cancer, and alzheimer's disease. In recent years the sphingolipid research community has sketched out the pathways through which these effects occur. With our knowledge of these pathways it is now time to begin filling in the details of these processes. This proposal will determine the folding motif, and binding sites for substrates and anionic phospholipids for neutral ceramidase and their relation to enzyme activty. Ceramidase hydrolyzes ceramide to sphingosine and a fatty acid. Ceramidases have been shown to exhibit the reverse, ceramide synthase, activity at lower pH's. The presence of another group of lipids, the anionic phospholipids have been shown to enhance ceramidase activity and are present in the sphingolipid enriched lipid micro-domains or rafts. This proposal will determine the binding sites of these two types of lipid. It will be achieved by firstly purifying the protein in quantities sufficient for structural studies. Crystallographic studies will be used to study complexes of the protein with ceramides and sphingosine to characterize the sphingolipid binding-site. Further studies, in conjunction with mutational analysis and activity assays will be used to characterize the anionic phospholipid-binding site. The protein ceramidase regulates the levels of ceramide available for cellular signaling. By understanding how ceramidase recognizes ceramide will enable highly specific inhibitors to be designed. The design of the inhibitors will then lead to therapeutics for medical intervention in cancer, alzheimer's disease and potentially lysozomal storage diseases.